Necessary and Insufficient: A Four-decade Search Points to a New Type of Gene Flaw

The history of science is filled with Eureka moments, sudden flashes of insight, often metaphorical, that tilt the prepared mind toward a new way of thinking, a solution to a seemingly unsolvable problem. The first such moment may have taken place more than 4,000 years ago, when the Greek mathematician and inventor Archimedes realized he could determine the volume of an oddly shaped object by measuring the amount of water it displaced. He was so thrilled that he leaped out of a public bath—where he had himself been displacing a volume of water—and ran home naked, shouting “Eureka” (I found it).

This is not one of those stories. This is a long steady slog, an ongoing series of small, painstaking, often conflicting discoveries, piling up one after another over 40 years, and still counting. It involves three generations of researchers, most of whom are currently working in the University of Chicago’s Knapp Center for Biomedical Discovery. It also took sustained support from the Cancer Research Foundation and others, plus hundreds of leukemia patients, as well as a few mice and flies. It was made possible by perseverance, rapidly evolving technology and a lot of detailed data collection and analysis. Although there has been huge progress, the story is not yet complete, and—it being winter in Chicago—no one has run home naked.

The tale began in late 1973 when Janet Rowley, at the time an associate professor of medicine, noticed something odd and quickly submitted a five-paragraph letter about it to the Lancet. She described two patients whose bone marrow cells, stained and examined through a microscope, showed “deficiencies” involving chromosome 7. Both had been treated for blood diseases that seemed to have recurred. “Whether the loss of part or all of chromosome 7 is fortuitous or not,” she wrote, “can only be determined by examination of additional patients.”

The search was on. By 1981, Rowley and clinical colleagues Richard Larson, Harvey Golomb and James Vardiman had medical histories and blood cells from 26 Chicago-area patients who developed acute myeloid leukemia (AML) a few years after previous treatment. Twenty of these patients had leukemic cells that had lost all or part of chromosome 7; many also had changes in chromosome 5. These therapy-related myeloid leukemias were troubling. The strong medicines used to treat the patients’ initial disease, often leukemia, seemed to have caused an even worse disease a few years later. This time, aggressive chemotherapy did not help; most patients died within a year.

By 1986, the effort to understand the genetic basis for this disease was being led by Michelle Le Beau, who had trained with Rowley. The team now had 62 such patients and had narrowed the search to the long arm of chromosome 7, known as 7q. They also investigated the specific therapies that triggered the genetic damage. About 75 percent had been treated with alkylating agents, a class of chemotherapy drugs that bind to DNA and interfere with replication.

At that time most genetic defects that were thought to cause cancer triggered rapid cell growth. Scientists had theorized that there might be other cancer-related genes called tumor suppressors that sinned by omission; they didn’t initiate the cancer but they failed to rein it in. In late 1986, a Boston-based research group described the first such tumor suppressor gene. That only made the search more difficult.

By the mid 1990s, new tools, such as fluorescent in situ hybridization, were helping Le Beau and colleagues narrow the focus, but progress remained incremental. Most of the tissue samples were missing the entire chromosome 7. However, the analysis of a small group of 81 patients with loss of only part of chromosome 7, known as a deletion, enabled them to narrow their focus. The emergence of distinct disease subtypes, which could be tied to different genes, complicated the problem. But by finding a few rare specimens with smaller deletions, they slowly sharpened the focus to a region known as 7q22.

This region still included more than two million base pairs, enough real estate to house 50 or more genes. Plus there was a new wrinkle. In no patient were both versions of a specific gene in the target region abnormal, which would be the hallmark of a tumor suppressor gene. There always seemed to be one functional copy.

Scientists were increasingly aware, however, that in some cases it might not require both copies of a tumor suppressor to fail. For some genes, loss of just one allele appeared to be damaging enough. The remaining copy could be normal but unable on its own to meet the demand, a state geneticists call haploinsufficiency.

“If discovering a ‘classic’ tumour-supressor gene is like finding a needle in a haystack,” Le Beau wrote in a “News and Views” article for Nature, “the challenge involved in uncovering haploinsufficient tumour-suppressor genes is akin to finding a specific piece of hay in a haystack.”

Pulling the right stalk from the haystack may be daunting, but in 2008 a team from the Broad Institute showed it was doable. They linked a different blood disease to haploinsufficiency of a gene on 5q, a chromosomal deletion first reported in 1974.

Megan McNerney

So in 2009, Meghan McNerney, a newly minted Pritzker MD/PhD doing a fellowship in molecular pathology and working in the lab of Kevin White in the University’s Institute for Genomics and Systems Biology, pulled together a team of collaborators to hone in on the chromosome-7 gene for therapy-related myeloid neoplasms.

By then they had higher resolution data from 35 therapy-related leukemia patients with chromosome 7 abnormalities. McNerney, White and other scientists in IGSB, combed through the suspect region from chromosome 7 to find candidate genes. One gene, once considered a likely prospect, particularly stood out.

CUX1 was within the region that was most often deleted and was known to regulate many other genes. In one patient sample, CUX1 was disrupted by a chromosome translocation. Although CUX1 is normally highly expressed in blood progenitor cells, the researchers found it was expressed in much lower, perhaps haploinsufficient, levels in many patient samples.

To confirm their suspicions, based on a series of genetic tests, White, McNerney and collaborators needed living, breathing evidence. So they created fruit flies—a novel model organism for leukemia—with only a single copy of the fly version of CUX1. When they took blood from the flies’ larvae, they found three times the normal levels of blood cells, a characteristic of therapy-related leukemia.

Next, working with pediatric stem cell transplant specialist John Cunningham, they developed mice in which the blood-forming cells were deficient in CUX1. These mice quickly developed features of leukemia.

Although there may be other genes in the commonly deleted sequence that play a role in disease pathogenesis,” the study authors wrote, “this is the first biological confirmation of a haploinsufficient myeloid tumor suppressor gene on chromosome band 7q22….Changes in CUX1 levels may have important phenotypes during hematopoiesis and deleterious consequences when altered.”

The trouble with tumor suppressor genes is that unlike oncogenes, which can provide a target for drug treatment, their absence or insufficiency makes a lousy treatment focus. This is “a good guy that’s gone,” McNerney said, not a bad boy to banish. “Gene therapy might be the best option.”

There may also be other ways to develop treatments based on this long-sought discovery, Le Beau said. If we learn how CUX1 is regulated “we could tweak its expression, or hit the pathway downstream, or find ways to influence its activity,” she said. “We may also find ways to use CUX1 testing to predict who is at risk prior to the initial treatment and to change the therapy for those patients.”

Despite the remaining challenges, this result is “really exciting,” Le Beau said. “This was a significant find and a great example of team work, a real group effort. We think this gene is a master regulator of development.” CUX1 mutations have also been found in lung and colon cancer.

After the paper was accepted by the journal Blood, McNerney met with Rowley to tell her that the team had finally bagged the chromosome-7, cancer-causing gene defect she had pointed to in 1973, one of her many seminal findings. This was a chance for Rowley to enjoy one more small celebratory moment in a Eureka-filled career. But on hearing the news, Rowley, instead of shouting “I found it,” began looking for her résumé and thumbing through the list of her early papers. Then she looked up at McNerney and asked: “I found that?”